Ribo zero magnetic kit

After behavioral test, mice were decapitated and the whole brain was taken out for quick dissection on ice. Striatal samples were sliced and punched to ensure equal amount of tissues. Midbrain samples were carefully isolated, and both samples were separated into the left and right, and were kept at −80 °C until use. Striatal DA content measurement was carried out in neurochemistry core in Vanderbilt University using HPLC. The total RNA were extracted from midbrain samples using Qiagen RNeasy mini kit (Cat No. 74104) and we sent 2.5 μg of each sample to Novogene, CA, USA for pair-end RNA sequencing using 250–300 bp insert cDNA library (rRNA depleted by Ribo-Zero^TM Magnetic Kit) on Illumina (CA, USA) HiSeq4000 platform. We applied FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality check and then STAR^{68 (link)} for alignment followed by edgeR package^{69 (link)} for differential gene expression analysis.

Expression of the TGF-β isoforms and their receptor genes were evaluated from global transcriptome RNAseq data. RNA was prepared from standard total RNA extraction with Trizol cleared of Ribosomal RNA using Ribo-Zero^™ Magnetic Kit from (Epicentre). Strand-specific RNAseq libraries were prepared with ScriptSeq^™ v2 RNA-Seq Library v2 Preparation Kit (Epicentre). Paired-end sequencing libraries were generated and sequenced using high-output kit version 2, HiSeq2000 platform, Illumina, USA. Finally, 60 plaques were used for differential gene expression analysis, and for Pearson correlation analysis between genes. A linear model was conducted to examine differentially expressed genes comparing patients with (Symptomatic) or without (Asymptomatic) symptomatic carotid disease. P-values were adjusted using the Benjamini & Hochberg method.

Total RNA was isolated from fungal and bacterial cells that were co-cultured for 8 h in six-well plates. For bacterial RNA isolation, the cells were disrupted using glass beads. For fungal RNA isolation, cells were frozen and homogenized using mortar and pestle, and then the total RNA was extracted using an RNA isolation kit (RNeasy Mini Kit; QIAGEN). Ribosomal RNA was depleted using Ribo-ZERO magnetic kit (Epicentre). The transcripts were fragmented and used as templates to generate strand-specific cDNA libraries by TruSeq Stranded Total RNA LT Sample Prep kit (Illumina). Each sample was sequenced using 100-bp paired-end reads on an Illumina HiSeq 2500 instrument. Macrogen Inc. supported library preparation, sequencing and partial data analysis. The reads were mapped to reference genomes of B. subtilis NCIB3610 (CP020102.1) or A. nidulans TN02A3 (GCA_000149205.1) with Bowtie 2 aligner. Read count per gene was extracted from known gene annotations with HTSeq program. After log₂ transformation of RPKM+1 and quantile normalization, differentially expressed genes were selected on conditions of log₂ > 2 in expression level.

To construct libraries, total RNA was extracted from leaves and roots of seedlings grown in different solutions (osmotic stress, salt stress and control) using the Trizol (Invitrogen) according to the manufacturer’s protocols. Ribosome RNA of six RNA samples was removed using Ribo-Zero™ Magnetic Kit (Epicentre). Thereafter the strand-specific sequencing libraries were constructed following a previously described protocol [68 (link)]. The paired-end sequencing (2 × 100 bp) was performed on an Illumina Hiseq2000 sequencer at the LC Biotech, Hangzhou, China.

We performed sequencing of the mRNA and lncRNA as described previously (Zheng et al., 2016 (link); Zheng & Jia, 2016 (link)). Briefly, we extracted the RNA of the PDLSCs using the Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). After digestion with DNase, we depleted rRNA using a Ribo-Zero magnetic kit and prepared a library according to the manufacturer’s instructions (Epicentre Biotechnologies, Madison, WI, USA). We performed paired-end sequencing using the Illumina Hiseq X Ten. After removing low-quality reads, we trimmed the raw reads and mapped them to the human genome (hg19) using TopHat2. We used HTseq to count the genes and calculated the reads per kilobase transcriptome per million mapped reads (RPKM) to evaluate the gene expression level. We used the EB-Seq package for the differential gene expression analysis among PDLSCs at each time point. We defined the differentially expressed genes (DEGs) based on fold changes greater than or equal to 2.0 and a false discovery rate of less than 0.05. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation Visualization and Integrated Discovery. Raw data of the performed RNA-seq have been recorded in the GEO public database (accession number: GSE99958).

Total RNA was extracted from the root tissue using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. rRNA was removed using a Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, USA). Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP, and buffer. cDNA fragments were purified using a QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands) and then ligated to Illumina sequencing adapters. Ligation products were subjected to size selection by agarose gel electrophoresis. Products were then amplified using PCR and sequenced using an Illumina HiSeq2500 platform. Three biological replicates were performed for each treatment.